Application of humic acid and biofertilizers changes oil and phenolic compounds of fennel and fenugreek in intercropping systems

The study investigated the effect of organic/biofertilizers in intercropping patterns on seed yield and yield components and essential oil, fatty acid, and phenolic compounds of fennel (Foeniculum vulgare L.) and fenugreek (Trigonella foenum-graecum L.). Experimental treatments included the application of humic acid (HA), biofertilizers (BFS), and the unfertilized control in five planting patterns [1 row fennel + 2 rows fenugreek intercropping (1F:2FG), 2 rows fennel + 2 rows fenugreek intercropping (2F:2FG), 2 rows fennel + 4 rows fenugreek intercropping (2F:4FG), and sole cropping of each species]. Sole cropping with BFS produced the highest seed yields for fennel (2233 kg ha−1) and fenugreek (1240 kg ha–1). In contrast, the 2F:2FG intercropping ratio with BFS yielded the maximum fixed oil content for fennel (17.4%) and fenugreek (8.3%). Application of HA and BFS enhanced oil yields by 66% and 75% in fennel and 40% and 57% in fenugreek, respectively. The 2F:2FG intercropping ratio with BFS produced the maximum essential oil constituents [(E)-anethole, estragole, and fenchone] in fennel. In addition, 2F:4FG with BFS and 1F:1FG with HA produced the highest unsaturated fatty acid (oleic and linoleic acids) concentration in both species. The 2F:2FG intercropping ratio with BFS and HA produced the highest chlorogenic acid and quercetin contents, respectively, in fennel. In contrast, the 2F:4FG intercropping ratio with HA produced the highest chlorogenic acid and caffeic acid contents in fenugreek. Intercropping fennel/fenugreek with BFS or HA improved the essential oil content (fennel only), fixed oil quality and quantity, and phenolic compounds and created a more sustainable cultivation system than sole cropping systems for both species under low-input conditions.


Materials and methods
Experimental site. The study was conducted as a factorial experiment with a randomized complete block design (RCBD) and three replications during the 2019 growing season at a farm (Long. 45° 44′ 19'' E., Lat. 36° 48′ 47'' N.) in Mahabad city in the Western Azerbaijan province, Iran. Weather data ( Table 1) was collected from the Iran Meteorological Organization (https:// www. irimo. ir/ eng/ index. php). The mean annual temperature and annual accumulated precipitation are 12 °C and 390 mm, respectively, and the elevation is 1320 m above sea level.
Treatments, land preparation, and cultivation. Prior to the study commencing, four baseline soil samples (0-30 cm depth) were taken across the experimental area to determine selected soil physicochemical properties ( Table 1).
Seeds used in the HA fertilizer treatment were first primed in HA solution. The HA compound used in this treatment contained 62% humic acid, 8% folic acid, and 10% potassium 8 . In the BFS fertilizer treatment, fennel and fenugreek seeds were uniformly sprayed and thoroughly mixed in the shade with BF (10 8 active bacteria per g BF) solution diluted in water 22. The HA and BFS were provided by Green Biotech Company Manufacturing, Qom, Iran.
Following the seed treatments for the HA and BFS fertilizer treatments, seeds of both plant species were air-dried at room temperature for one day and then sown on March 18, 2019. The plants were irrigated with 10 L ha -1 at the stem elongation and flowering stages, with the weeds removed by hand as required. No synthetic fertilizers were used in this study.
The plant material and seeds were obtained under the supervision and permission of Urmia University and according to national guidelines; all authors complied with local and national guidelines.

Measurements.
Fenugreek and fennel were harvested at the end of the growing season, on July 19, 2019 and September 5, 2019, respectively, when about 75% of the pods were yellowed. At this time, 10 plants were randomly selected from 3.2 m 2 (2 m long × 1.6 m wide) across the four central rows in each plot to determine yield components, including plant height, pods per plant, seeds per pod, and 1000-seed weight for fenugreek and plant height, umbels per plant and 1000-seed weight for fennel.
Fennel essential oil extraction and analysis. The EO of fennel plants was extracted by hydrodistillation in a Clevenger. For this purpose, 30 g of dried seed samples were weighed from each plot and ground to pass through a 1-mm screen. Ground samples were then placed in a jar with 300 mL water and boiled inside the Clevenger for 3 h to extract the essential oil. The extracted EO was weighed (g) and the EO content and EO yield were calculated as follows 10 : In addition, the EO yield of fennel (kg ha -1 ) was calculated by multiplying of EO content (%) and seed yield of fennel. Extracted EOs were dried using anhydrous sodium sulfate and stored at 4 °C until analysis. Fixed oil extraction. Briefly, 5 g ground seed samples of fennel and fenugreek for each treatment were mixed in 300 mL n-hexane to extract the fixed oil in a Soxhlet apparatus. After 6 h of extraction, the solvent was removed from the oil by rotary evaporation. The extracted oil was stored in amber glass bottles at 4 °C until the oil constituents were analyzed by GC-MS 10 . In addition, the oil yield of two plants was calculated by multiplying the oil content (%) and seed yield of fennel/fenugreek. Method of extraction of phenolic acids. Dried seeds were dissolved in 2 mL of 80% MeOH and then transferred to an ultrasonic bath for 30 min. Next, the homogenates were centrifuged at 3,000 rpm for 15 min and transferred to sealed jars. Extracts were crushed through fine membrane lighters and then stored at 20 °C. Finally, 20 mL of the extract was injected into an HPLC to determine separation and analysis of phenolic acids.
Isolation, identification, and determination of phenolic acid quantities. Analysis of phenolic acids was performed using an Agilent 1100 (HPLC) comprising 20 μL injection loop, degasser, diode-array detector (HPLC-DAD) adjusted at 250, 272, and 310 nm, four-solvent gradient pump, Octadecylsilane column, and Chemstation software for data processing. To isolate the compounds, the elution process was applied as follows: mobile phase initiated with 10% acetonitrile and 90% acetic acid (1% solution) at a flow rate of 1 mL/min, to reach 25% acetonitrile and 75% acetic acid, and 65% acetonitrile and 35% acetic acid at a flow rate of 1 mL/ min after 10 min. The isolation time was 15 min.
Land equivalent ratio (LER). The partial LER of fennel (LER F ) and fenugreek (LER FG ) and total LER (LER T ) were calculated as follows 30 : Statistical analyses. Analysis of variance and mean comparisons were performed with Duncan's multiple range test at the P < 0.05 level using the SAS 9.4 software package to assess the impact of intercropping patterns and fertilizer sources on agronomic variables and yield in fennel and fenugreek, essential oil productivity of fennel, and oil content of both species. The intercropping patterns, fertilizer sources, and their interaction were considered fixed effects, while blocks were considered random effects. All graphs were drawn in MS-Excel.

Results
Fennel. The main effect of fertilization (F) significantly impacted all measured parameters of fennel. Intercropping (I) pattern affected all parameters except plant height and 1000-seed weight. Significant I × F interactions occurred for umbel number, seed yield, essential oil content (EO), EO yield, oil content, and oil yield (  1A). Compared to the unfertilized control, HA and BFS increased fennel plant height by 10% and 13%, respectively (Fig. 1B).
Essential oil content and yield. The different intercropping patterns produced higher EO contents of fennel than fennel sole cropping. The highest absolute EO content of fennel (4.22%) occurred in 2F:2FG fertilized with BFS, although this did not statistically differ from the 2F:2FG fertilized with HA (4.04%) or 2F:4FG fertilized with HA or BFS (3.8% and 4.00%, respectively) (Fig. 3A). The lowest EO contents occurred in the unfertilized control (2.38%), HA (2.55%), and BFS (2.57%) in the Fs system. Averaged across fertilization treatments, the EO content of fennel in 1F:2FG, 2F:2FG, and 2F:4FG increased by 36%, 52%, and 44% compared to fennel sole cropping, respectively. Within each intercropping pattern, and with the exception of Fs, the HA and BFS treatments had higher EO contents of fennel, none of which significantly differed, increasing by 25% and 29%, respectively (Fig. 3A).
Phenolic compounds. The main phenolic compounds of fennel were chlorogenic acid (10.4-15.3 ppm), quercetin (7.0-17.2 ppm), and cinnamic acid (4.1-8.9 ppm). The highest chlorogenic acid and quercetin contents occurred in 2F:2FG fertilized with BFS and HA, respectively, while the lowest contents occurred in the fennel sole cropping system without fertilizer. Averaged across the three intercropping patterns, the chlorogenic acid and quercetin contents were 18.5% and 80.1% higher than the fennel sole cropping system. The chlorogenic acid and quercetin contents increased by 13% and 17% with BFS and 22% and 15% with HA, respectively (Table 5).
Fenugreek. The main effects of intercropping (I) pattern (C) and fertilizer (F) were significant for all parameters analyzed in fenugreek. Significant I × F interactions occurred for plant height, pod number per plant, seed yield, oil content, and oil yield of fenugreek (Table 6).   (Fig. 4B).
Seed yield. Means comparisons showed that sole cropping produced higher fenugreek seed yields than intercropping patterns. Sole cropping with BFS (1240 kg ha -1 ) and HA (1217 kg ha -1 ) produced the highest seed yields followed by the unfertilized control (Fig. 6A). The unfertilized control in 1F:2FG (437 kg ha -1 ) and 2F:2FG (467 kg ha -1 ) produced the lowest fenugreek seed yields. In all cases, and within each cropping pattern, BFS and HS produced higher fenugreek seed yields than the unfertilized control. As a result, BFS and HA increased fenugreek seed yield by 25.2% and 31.5% compared with the unfertilized control, respectively (Fig. 6A). Table 6. Analysis of variance for the effects of cropping patterns and fertilization on evaluated traits in fenugreek. NS, *, and ** indicated non-significance differences and significant differences at the 5% and 1% probability levels, respectively. www.nature.com/scientificreports/ Oil content and yield. The 2F:2FG cropping pattern with BFS produced the highest fenugreek oil content (8.3%), while the unfertilized control in sole cropping produced the lowest (5.9%). Across fertilizer treatments, 1F:2FG, 2F:2 FG, and 2F:4 FG produced 11.7%, 18.5%, and 15.7% higher fenugreek oil contents than sole cropping, respectively. In the 2F:2FG and 2F:4FG cropping patterns, BFS produced higher oil content (%) than HA. As a result, across cropping patterns, HA and BFS increased fenugreek oil content by 12.3% and 19.4%, respectively (Fig. 6B). Sole cropping with HA and BFS and 2F:2FG with BFS produced the highest fenugreek oil yields (77.1, 80.0, and 74.4 kg ha -1 , respectively), while the unfertilized controls in 1F:2FG and 2F:4FG produced the lowest (27.51 and 29.8 kg ha -1 , respectively). The 1F:2FG, 2F:2FG, and 2F:4FG cropping patterns produced 45.9%, 20.7%, and 41.5% lower fenugreek oil yields than fenugreek sole cropping, respectively. Moreover, except for sole cropping, BFS produced the highest fenugreek oil yield, followed by HA and the unfertilized control (Fig. 6C).

Discussion
Application of humic and bio-acid fertilizers in fenugreek and fennel intercropping can improve the quantitative and qualitative yield of both species. In our field study, we calculated higher land equivalent ratios for both crops in the intercropping systems than the sole cropping systems. In our study, sole cropping produced higher growth parameter and productivity values for both plant species than intercropping. Therefore, a reduction in the values of most growth parameters and in the partial productivity of plants in intercrops could be explained by a lower number of partial plants density of each species in intercropping patterns in comparison with sole cropping systems 31 . However, comparing partial yields in sole cropping with those under intercropping does not explain the total productivity in each system. For this purpose, the LER index is a better indicator of productivity level. Parallel with our hypothesis, we calculated LER values > 1 in all intercropping patterns except for 1F:1FG without fertilization, indicating that fennel with fenugreek intercropping enhanced total system productivity compared with sole cropping. In addition, intercropping produced 6-42% higher LER values than sole cropping, implying higher productivity of both species under intercropping than sole cropping in most cases. In other words, 6-42% more land area would be needed under sole cropping to achieve the same yields under intercropping. Most intercropping patterns had partial LER values > 0.5, further highlighting the superior productivity under intercropping based on land use efficiency 32 . In sole cropping conditions, the higher intraspecific competition decreased the nutrient use efficiency of nutrients. Therefore, it can be concluded that the higher nutrient accessibility by BFS application along with improvement of environmental use efficiency and better spatial, temporal and chemical complementarity of both plants in intercropping patterns enhanced the LER index when compared with sole cropping conditions 33 . Moreover, atmospheric nitrogen fixation by the legume component of the system (fenugreek) and its direct/indirect transfer to the non-legume component (fennel) reduced the competition for inorganic nitrogen at the whole system level and enhanced plant productivity by improving nutrient absorption. Liu et al. 34 noted that intercropping legume/non-legume species increased legume nodule formation and fixation due to the stimulation of nitrogen fixation by the non-legume and dissolution of P that acidifies the rhizosphere.
Intercropping patterns with BFS and HA generally produced higher plant productivity and LER index than unfertilized intercropping due to the positive role of BFS and HA fertilizers as nutrient suppliers for plants 20 . In line with our second hypothesis, BFS and HA improved plant growth characteristics by solubilizing and fixing nutrients for easier plant uptake, regulating hormones, and exuding plant growth regulators and phytohormones (e.g., IAA, cytokinins, GA, and ethylene) 35 . Rezaei-Chiyaneh et al. 10 reported that BFS increased nodule number and dry weight, nitrogen fixation, and overall plant productivity in common bean. They also noted that BFS increased several plant growth parameters by reducing soil pH and improving the conditions for plant nutrient uptake. Similarly, Faridvand et al. 14 showed that different intercropping patterns of Moldavian balm (Dracocephalum moldavica L.) with mung bean (Vigna radiata L.) had higher LER indexes than sole cropping of each species.  www.nature.com/scientificreports/ Application of BFS and HA significantly increased the seed weight of fennel and fenugreek. Similarly, Kumari et al. 36 reported that BFS application significantly increased the seed weight of bell pepper (Capsicum annuum L.). Studies have shown that essential oil and fixed oil contents in fennel and fenugreek significantly correlate with seed yield 23 . Equally, the highest EO and fixed oil yields for both species in our study occurred in the 2F:2FG intercropping pattern treated with BFS, which had the highest EO and fixed oil contents. Consequently, any factor that increases these indices may also increase the oil yield.
In MAPs, essential oils are final terpenoid products formed by a large group of enzymes known as terpene synthases, derived from a basic structure of five carbons (C 5 H 8 ), commonly called an isoprene unit, and classified depending on the number of these units in its skeleton. Biosynthesis of EO depends on the presence of different input substances and enzymes. Anethole, a monoterpene position isomer, is the main constituent of essential oils from aromatic plants, including anise, star anise, and fennel. Anethole is used as a flavoring agent in the food and pharmaceutical industries in the United States and many other countries 37 . In our study, the 2F:2FG intercropping ratio with BFS produced the highest percentage of (E)-anethole.
Calsamiglia et al. 38 noted that glucose availability in plant cells, produced during photosynthesis, plays an important role in increasing terpenoid constituents in MAPs. Rostaei et al. 39 noted that nutrient accessibility, especially N and P, in MAPs plays a key role in the development and division of cells containing EO, EO channels, glandular trichomes, and secretory ducts. Similarly, consistent with our hypothesis, Nurzynska-Wierdak et al. 40 found that fertilizers can significantly modify the EO content and chemical constituents of MAPs, most likely associated with changes in synthesis pathways and the role of these components in plant physiology. Therefore, the higher EO content and constituents of fennel supplied with BFS in intercropping system may be due to the enhanced nutrient availability promoting enzyme activity and precursor compounds of EOs, including isoprene, phenylpropanes, and others 40,41 . Overall, EO quantity and quality of fennel improved in intercropping patterns treated with BFS. Similarly, Rezaei-Chiyaneh et al. 10 showed that fennel/common bean intercropping with BFS improved EO content and chemical composition in terms of increased (E)-anethole, fenchone, and limonene concentrations.
In our experiment, BFS and HA increased the unsaturated fatty acid structure (FAs) and thus oil quality in most intercropping treatments. The ratio of unsaturated to saturated FAs is an important index for determining oil quality. As hypothesized, the increased concentration of unsaturated FAs in intercropping patterns treated with BFS suggests that appropriate inoculations can further enhance the health benefits of fixed oil in both fennel and fenugreek, making the seed oil more appropriate for human consumption. Likely, the higher nutrient availability in the HA and BFS treatments due to plant-growth-promoting microorganisms and atmospheric N fixation increased the photosynthetic rates of both plant species. As a result, the biosynthetic cycle of FAs may have been accelerated due to the higher supply of carbon resources (especially citrate content) and other precursor compounds, including ATP and NADPH, eventually increasing FA production 42 .
Our results showed that the phenolic compounds of fennel and fenugreek significantly increased in the 2F:2FG and 2F:4FG intercropping patterns treated with HA and BFS. In line with our second hypothesis, the biosynthesis of phenolic compounds can positively influence nutrient availability, especially N 43 . Kováčik et al. 44 reported that phenylalanine ammonia-lyase is involved in the biosynthesis of phenolic compounds and regulated by N availability. Therefore, the higher phenolic compounds in the abovementioned intercropping treatments could be due to higher nutrient availability, with the nitrogen fixed by the legume species being transferred to the non-legume companion plants. In addition, the higher concentrations of phenolic compounds under HA and BFS were attributed to the improved nutrient availability, increasing the activity of enzymes such as phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4coumaryl-CoA (4CL), and phenolic contents 45 . Thus, bio/organic fertilizer application in the intercropping systems investigated in this study positively affected the formation of phenolic compounds, which could improve the overall defense mechanism of competing plants in intercropping systems.

Conclusion
Biofertilizer and humic acid applications significantly increased fennel and fenugreek yields. of The 2F:2FG (fennel:fenugreek) intercropping ratio treated with biofertilizer was the best option in most comparisons and had the highest total land equivalent ratio index. Biofertilizer and humic acid applications increased essential oil content and essential oil yield of fennel and fixed oil content and fixed oil yield of fennel and fenugreek compared to the unfertilized control. Moreover, different intercropping patterns of fennel/fenugreek treated with biofertilizer and humic acid improved the essential oil quality of fennel and the fixed oil quality and phenolic compounds of both species. We conclude that the 2F:2FG and 2F:4FG intercropping patterns treated with biofertilizers and humic acid are excellent options for farmers looking for cleaner and more eco-friendly strategies to increase their income by improving essential oil and fixed oil quality in intercropping systems.